Current Projects

NSF and SNARE complex disassembly

A model protein disassembly reaction NSF was the first AAA+ protein implicated in membrane trafficking. Its primary function is to disassemble complexes of membrane-bound SNARE proteins after they come together to promote membrane fusion. We are currently using novel in vitro assays to define how NSF and its cofactor alpha-SNAP recognize and operate on different SNARE complex substrates and are regulated by interacting proteins. We are also interested in further understanding NSF’s role in modulating other membrane-based protein complexes.

VPS4/SKD1 and the control of multivesicular body biogenesis

We are interested in understanding the mechanism by which cells internalize membrane into endosomes to form multivesicular bodies (MVBs). Internalization of membrane and associated cargo into the MVB is essential for digestion and degradation in the lysosome, and is also used by specialized cells to generate exosomes. A set of eighteen “class E” proteins conserved from yeast to mammals is thought to control MVB biogenesis. Some or possibly all of these proteins are also used by nonlytic viruses such as HIV to bud from the plasma membrane of infected cells in a process that is topologically equivalent to budding into the endosomal lumen. How membrane invagination and vesicle fission into the MVB or away from the plasma membrane are controlled is not known. The AAA+ protein VPS4/SKD1 is essential in this pathway, and interacts with a set of “endosomal complex required for sorting” (ESCRT) proteins known as the ESCRT-III proteins. We are studying the structure, function, and interactions among these proteins in order to define their contribution to MVB biogenesis. We hypothesize that regulated assembly and disassembly of ESCRT-III polymers is directly responsible for membrane invagination into the MVB.

TorsinA and the nuclear envelope

Early-onset (DYT1) torsion dystonia is a CNS-based movement disorder usually associated with a single amino acid deletion (?E302/303) in the protein torsinA. TorsinA is an AAA+ ATPase localized to the endoplasmic reticulum in higher eukaryotes. Based on the localization of ATPase defective “substrate trap” mutants, we and others have defined the nuclear envelope as the likely site of torsinA action and hypothesize that the enzyme may play a role in regulating connections between inner and outer nuclear membranes. How deficiencies in this ubiquitously expressed enzyme lead to the neuronal dysfunction underlying torsion dystonia is a mystery that promises to shed light on the interplay between nuclear envelope and disease. We are working to define the cellular pathway(s) in which torsinA operates in order to define the normal function of this unique family of ER-lumenal AAA+ proteins. We also want to understand how disease-associated mutations in torsinA alter the protein’s structure and/or function and contribute to the development of disease.